Impact of macromolecular crowding on RNA/spermine complex coacervation and oligonucleotide compartmentalization†,Soft Matter

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Impact of macromolecular crowding on RNA/spermine complex coacervation and oligonucleotide compartmentalization†
Soft Matter ( IF 2.9 ) Pub Date : 2017-12-15 00:00:00 , DOI: 10.1039/c7sm02146a
A. M. Marianelli _{1,

2,

3,

4} , B. M. Miller _{1,

2,

3,

4} , C. D. Keating _{1,

2,

3,

4}

Affiliation

We report the effect of neutral macromolecular crowders poly(ethylene glycol) (PEG) (8 kDa) and Ficoll (70 kDa) on liquid–liquid phase separation in a polyuridylic acid (polyU)/spermine complex coacervate system. The addition of PEG decreased both the amount of spermine required for phase separation and the coacervation temperature (T_C). We interpret these effects on phase behavior as arising due to excluded volume and preferential interactions on both the secondary structure/condensation of spermine-associated polyU molecules and on the association of soluble polyU/spermine polyelectrolyte complexes to form coacervate droplets. Examination of coacervates formed in the presence of fluorescently-labeled PEG or Ficoll crowders indicated that Ficoll is accumulated while PEG is excluded from the coacervate phase, which provides further insight into the differences in phase behavior. Crowding agents impact distribution of a biomolecular solute: partitioning of a fluorescently-labeled U15 RNA oligomer into the polyU/spermine coacervates was increased approximately two-fold by 20 wt% Ficoll 70 kDa and by more than two orders of magnitude by 20 wt% PEG 8 kDa. The volume of the coacervate phase decreased in the presence of crowder relative to a dilute buffer solution. These findings indicate that potential impacts of macromolecular crowding on phase behavior and solute partitioning should be considered in model systems for intracellular membraneless organelles.

中文翻译：

大分子拥挤对RNA /精胺复合物凝聚和寡核苷酸区室化的影响†

我们报告了中性大分子拥挤剂聚乙二醇（PEG）（8 kDa）和Ficoll（70 kDa）对聚尿苷酸（polyU）/精胺复杂凝聚层系统中液相分离的影响。PEG的添加减少了相分离所需的精胺量和凝聚温度（T _C）。我们将这些对相行为的影响解释为由于精子相关的polyU分子的二级结构/缩合以及可溶性polyU /精胺的聚电解质复合物缔合而形成的凝聚液滴的排除体积和优先相互作用所引起的相行为。在荧光标记的PEG或Ficoll拥挤剂的存在下形成的凝聚层的检测表明，聚凝聚层中聚集了Ficoll，而PEG从凝聚层相中排除，这提供了对相行为差异的进一步了解。拥挤剂影响生物分子溶质的分布：荧光标记的U15 RNA寡聚体在polyU /精胺凝聚层中的分配增加了约20倍，其中Ficoll 70 kDa增加了两倍，而20重量％PEG增加了超过两个数量级。 8 kDa。相对于稀缓冲液，在拥挤的情况下凝聚相的体积减小。这些发现表明，在细胞内无膜细胞器的模型系统中，应考虑大分子拥挤对相行为和溶质分配的潜在影响。

更新日期：2017-12-15

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